Investigating factors of metabolic bone disease in baboons (Papio spp.) using museum collections

Abstract Objectives To assess manifestations of metabolic bone disease (MBD) and their potential environmental and phenotypic factors in captive and non‐captive baboon (Papio spp.) specimens. Materials and methods Our sample consisted of 160 baboon specimens at the Smithsonian's National Museum of Natural History accessioned from 1890 to 1971. Combining cranial indicators of MBD and the museum's historical data, we examined factors contributing to likely instances of MBD. We used binomial‐family generalized linear models to assess differences in MBD frequency by environment (captive, non‐captive), specimen accession year, and skin color (light, medium, dark). Results Indicators of MBD were most frequently observed in captive baboons, with a decrease in MBD frequency over time. Fifteen non‐captive individuals showed indicators of MBD, which are the first published cases of MBD in non‐captive nonhuman primates (NHPs) to our knowledge. The most common MBD indicators were bone porosity (n = 35) and bone thickening/enlargement (n = 35). Fibrous osteodystrophy was observed frequently in our sample, likely relating to nutritional deficiencies. We found no association between exposed facial skin color variation and MBD. Conclusions Our findings are consistent with historical accounts of MBD prevalence in captive facilities, especially earlier in the 20th century. A decrease in MBD prevalence later in the 20th century likely reflects improvements in housing, diet, and veterinary care in captive settings. Causes of MBD development in non‐captive baboons should be further explored, as understanding the potential health impacts that anthropogenic environments impose on NHPs is imperative as humans increasingly alter the natural world in the 21st century.

The general diagnosis of "metabolic bone disease" is commonly used instead of specific diagnoses (such as rickets, osteomalacia, or fibrous osteodystrophy) or when multiple conditions are present in an individual (Uhl, 2018). Of all MBDs, fibrous osteodystrophy appears most prominently in the cranium and mandible, whereas rickets and osteomalacia are better detected through postcranial elements (Table 1).
The bilateral enlargement of fibrous osteodystrophy lesions in the cranium and mandible is a direct result of osteoclastic bone resorption; bone mineral (calcium and phosphorus) is transferred back to the circulatory system, and distortion of the remaining cancellous bone occurs (Canington & Hunt, 2016;Olson et al., 2015). Rickets represents a softening of the bones linked to vitamin D deficiency during development, which occurs prior to epiphyseal fusion (called osteomalacia during adulthood after epiphyseal fusion has ceased; Wharton & Bishop, 2003).
While genetic factors may play a role in MBD etiology, nongenetic factors are most often implicated (Farrell et al., 2015;Hannan et al., 2019;Uhl, 2018). MBD presence in animals can be particularly revealing about their environmental conditions during life, including anthropogenic (human constructed or influenced) spaces. Many animals are not equipped to handle the novel, rapidly imposed pressures of some anthropogenic environments, which can alter diet, mobility, social behavior, and reproduction (e.g., primates and carnivores; Michalski & Peres, 2005). MBDs are rarely recorded in non-captive (i.e., "wild") populations, although nutritionally related MBD has been reported in wild birds, possibly due to a calcium-deficient diet related to range expansion into suboptimal anthropogenic habitats (Cousquer et al., 2007;Phalen et al., 2005). MBD is well recognized in many domesticated animals (including sheep, goats, llamas, alpacas, cattle, pigs, horses, reptiles, cats, and dogs; Dittmer & Thompson, 2011), frequently linked to deficiencies in nutrition and/or sunlight exposure (Dittmer & Thompson, 2011;Uhl, 2018). For animals in captive settings, including zoological parks and research facilities, MBD has long been associated with nutritional and environmental stress (Fiennes, 1974;Mann, 1930;Ratcliffe, 1966;Wackernagel, 1966).
Historical reports of MBD are confounded by a lack of pathophysiological knowledge and consensus on diagnosing zoo animal pathologies. Instead, all MBDs were broadly classified as "rickets." At the London Zoo in 1890, surgeon John Bland-Sutton noted that "half the monkeys and lemurs brought to this country die rickety" (Bland-Sutton, 1895:266). However, the description of "rickets" in the historical sense more broadly encompassed a suite of MBDs, rather than the specific diagnosis used today. Some historical cases, like "rickets" in lion cubs at the London Zoo in 1889, were most likely fibrous osteodystrophy, where lesions form by bone resorption and replacement with immature bone that lacks proper structure and mineralization (Table 1; Chesney & Hedberg, 2010).
While misdiagnoses were common, historical reports also highlight efforts to change zoological practices for disease prevention; in T A B L E 1 Definitions of specific MBD conditions that may be evident in the study sample, following Canington and Hunt (2016), Craig et al. (2016), Fiennes (1974), Farrell et al. (2015), and Uhl (2018 which reversed MBD in these animals (Chesney & Hedberg, 2010). By the 1930s, diet and sunlight had been identified as the key to disease prevention in captive settings (Fiennes, 1974;Mann, 1930;Ratcliffe, 1966;Wackernagel, 1966). As enclosures shifted from restrictive cages to naturalistic designs, zoo veterinarians began to remedy the high prevalence and severity of MBD through dietary means such as vitamin D, calcium, and phosphorus supplements, as well as UV light therapy to produce cutaneous vitamin D (Fiennes, 1974;Ratcliffe, 1966;Wackernagel, 1966). Since animal welfare legislation was enacted in the mid-20th century (Hosey et al., 2009), and alongside greatly improved diets, habitats, and veterinary care practices (Fiennes, 1974;Gutierrez et al., 2021;Smithsonian Institution, 1942, 1952, 1957, MBDs have been greatly reduced in captive NHPs, save a few isolated cases (Hatt & Sainsbury, 1998;Morrisey et al., 1995;Wolfensohn, 2003).  (Farrell et al., 2015), although the "natural baseline" of MBD in non-captive populations is unknown. Notably, baboons display strong interspecies variability in skin and pelage morphologies, including pigmentation (Hill, 1967;Kamilar, 2006). In both non-captive and captive living baboons, exposed skin color influences vitamin D 3 production, but not downstream metabolism (Ziegler et al., 2018). Because of the known connection between sunlight and MBD, it is plausible that skin color differences could lead to variable disease outcomes, as documented in humans (Holick, 2004). In addition, the relative availability of dietary vitamins and minerals, and/or the functionality of their physiological pathways, may also contribute to variable disease outcomes. As far as is known, all catarrhine primates, including Papio baboons, have similar requirements for vitamins and minerals (Milton, 2003), including calcium and phosphorus, and similar levels of circulating parathyroid hormone (Fincham et al., 1993), all of which can cause MBD if imbalances or disruption to physiological pathways occur. There are some physiological differences within the Order Primates specifically related to vitamin D, for example, differential synthesis rates between vitamin D 2 and vitamin D 3 and different levels of circulating vitamin D (Crissey et al., 1999;Gacad et al., 1992;Marx et al., 1989). These differences are most pronounced between platyrrhine and the vitamin D 3 -resistant catarrhine primates, the latter having very low vitamin D uptake by target cells (i.e., cell "resistance") resulting in higher levels of circulating bioactive vitamin D (Adams et al., 1985;Gacad et al., 1992).
In this study, we investigated cranial and mandibular indicators of MBD in baboons (Papio spp.) to better understand the potential health impacts of anthropogenic environments on NHPs. Past studies of MBD have predominantly focused on pathological analysis and approaches to remedying animal disease (e.g., Fiennes, 1974;Ratcliffe, 1966;Wackernagel, 1966). However, we take an ecological approach to the study of MBD, considering how diseases can arise when animals are subjected to environmental conditions that differ from their natural habitat (Eller et al., 2019). Using a large sample of non-captive and captive baboon cranial and mandibular specimens spanning nearly a century although the latter is not tested here.

| Sample
We macroscopically examined 160 baboon (Papio spp.) skulls in the Division of Mammals at the NMNH (Supplementary Table 1). Due to the over-representation of primate skulls in the NMNH collections relative to postcranial elements, we focused on cranial and mandibular manifestations of MBD to allow for a larger sample size. Table 2 summarizes the demographic information collected from specimen tags and NMNH accession records for each specimen. Taxonomic designations followed the Zinner et al. (2011) scheme, which include six distinct Papio species: P. anubis, P. papio, P. ursinus, P. cynocephalus, P. kindae, and P. hamadryas.
Environmental assignments were based on whether an individual died in their native African habitat ("non-captive") or in captivity ("captive"), including zoological parks and biomedical facilities. Non-captive individuals were further geographically and temporally subdivided to consider habitat diversity across Africa (Supplementary Table 2).
Additionally, the specimen's date of accession (the most reliable date available) was recorded from each tag, indicating the year that NMNH acquired the remains, usually soon after death (Supplementary Table 3).
Age was estimated using molar eruption stages following Kahumbu and Eley (1991)  Facial skin pigmentation was classified into three color groups: light, medium, and dark ( Figure 1). Following Ziegler et al.
(2018), three baboon species were categorized in their visually determined color scale: P. anubis as dark, P. cynocephalus and P. kindae as medium, and P. hamadryas as light. For P. papio and P. ursinus, we used Kingdon et al. (2013) for skin and pelage pigmentation descriptions to place P. papio in the "dark" category and P. ursinus in the "medium" category. When possible, associated specimen skins were visually assessed to confirm the group assignment.
T A B L E 2 Taxonomic distribution of specimens by skin color group, sex, age, accession year, and environment type Note: Specimens were classified as "captive" if the individual lived in a captivity at any point during life, regardless of birthplace. Accession years were grouped for descriptive purposes but not for statistical testing. Bone porosity in any of the following skull regions: sagittal suture, glabella, and maxilla often associated with pitting; mandible associated with swollen and spongy look; general increase in bone porosity Farrell et al., 2015 35 Bone thickening and enlargement in any of the following skull regions: frontal, parietal, and/or temporal bones of the cranium; zygomatic bones extending anteriorly into maxilla; maxilla with bilateral enlargement from alveolar margins to frontal process; mandible; nasal conchae; and palate Canington & Hunt, 2016;Farrell et al., 2015;Fiennes, 1974;Uhl, 2018 35 Mandibular condyles are underdeveloped and/or coronoid processes are missing Canington & Hunt, 2016;Farrell et al., 2015 10 Rounded orbital margins with softened orbital edge Farrell et al., 2015 8 Eruption and position of teeth affected when maxilla and mandible has swelled Farrell et al., 2015;Fiennes, 1974 4 General decrease in bone density Canington & Hunt, 2016;Farrell et al., 2015 3 Linear enamel hypoplasia in teeth Guatelli-Steinberg and Skinner, 2000 4 Fragile, friable, and/or brittle bone regions with inner trabeculae exposure in some cases the basis for our skeletal assessments (Canington & Hunt, 2016;Craig et al., 2016;Farrell et al., 2015;Fiennes, 1974;Uhl, 2018). Because of the longstanding confusion on specific MBD conditions, modern definitions relevant to the current study are provided in Table 1, following Craig et al. (2016). Although MBD does present postcranially, there are far fewer postcranial skeletons available for study at the NMNH (n = 9), and thus only cranial and mandibular evidence was considered. As MBD broadly encompasses a wide range of conditions, we identified a series of pathological criteria, any of which can be indicative of MBD by visual assessment (Figure 2; Table 3). MBD pathological designations were made by agreement of all authors.

| Statistical analysis
We used binomial-family generalized linear model (GLMs) to assess All analyses were conducted with the R statistical programming language (R Core Team, 2021). for Research and Education in San Antonio, Texas (n = 61; 69.3%).

Most of the captive baboons in our sample lived and died in the
One specimen came from the Oregon Regional Primate Center   (Figure 2; Table 3).
Of the 51 pathological individuals, 14 died in their native habitats 0.14; Figure 3b). The 51 specimens with MBD pathologies represent five of the six species, excluding P. kindae, and all three categories of exposed facial skin pigmentation (Figure 1). Facial skin color did not significantly predict MBD frequency (LRT: χ 2 = 5.6, p = 0.06). MBD was not successively more frequent among the "medium" and "dark" groups as compared to the "light" group (Table 4). In the three statistical models examining MBD frequency by environment, specimen accession year, and skin color, age was a significant predictor of MBD (χ 2 = 10.3-12.9, p < 0.05; Table 5), while sex (Table 6) and species designation were not.

| MBD indicators and disease specifications
The most frequently identified pathology within our sample was the enlargement and thickening of cranial and mandibular regions. Many of these lesions are consistent with skeletal manifestations of fibrous osteodystrophy, formed by calcium and phosphorus imbalance ( Figure   2; Table 1; Bandarra et al., 2011;Canington & Hunt, 2016;Craig et al., 2016;Long et al., 1975;Lynch et al., 1999;Olson et al., 2015). social groups, sometimes even grouping multiple species from different habitats together, to cater to the public for the purposes of public recreation and science communication (Figure 4). Possibly for this reason, nearly all specimens derived from the NZP showed evidence of MBD, while very few from the Southwest Foundation did. A rich record of NZP documents from the NMNH collections databases and Smithsonian Institution Archives supports the cranial evidence. These records reveal malnutrition, inadequate veterinary care, and poor housing conditions though the mid-20th century, all which may have contributed to the high prevalence in MBD (Gutierrez et al., 2021).
A complementary explanation is that captive animal care practices had improved by the time the Southwest Foundation opened in 1960.
MBD was very common in captive NZP individuals that were accessioned into the NMNH collection earlier in the 20th century, but the rate of MBD prevalence decreased in the subsequent decades with the inclusion of additional Southwest Foundation specimens. It is possible that with better animal welfare practices, captive animals experienced healthier environments later in the 20th century ( Figure 5). Our results showed that differences between captive and non-captive baboon populations became nonexistent into the 1960s and 1970s, a pattern driven by decreasing disease prevalence in the captive group over time. Within the non-captive group, there was no evidence for a temporal change in MBD frequency.
To our knowledge, this study is the first to identify MBD in noncaptive NHPs. We identified a total of 14 non-captive baboon specimens with evidence of MBD, spanning 1905 to 1967 (Supplementary Table 1). This result is interesting, but at this time we cannot do more than speculate as to the cause. Under wild conditions, baboons are susceptible to MBD likely through an under-nourishing diet or possibly through acquired gastrointestinal parasites that alter nutrient uptake (as seen in non-primate vertebrates; Loukopoulos et al., 2007;Lynch et al., 1999). It is also possible that non-captive baboons are still experiencing environments which are not entirely free from human influence, leading to anthropogenic changes in food availability  analyzed 875 NHP specimens in the NMNH collections and found that over 90% of the NHP specimens do not come from socalled "wild" settings as often assumed. Instead, these animals lived in habitats within terrestrial biomes of varying degrees of anthropogenic F I G U R E 4 Interior view of the monkey house at the National Zoological Park in 1910(Smithsonian Institution Archives, 1910. Enclosures consist of small cages with little natural sunlight, no greenery, no temperature control, and no animal enrichment. In the following years, all primates were moved to a new monkey house that was cleaner and had better ventilation, but it was not until the 1950s that better funding allowed for greatly improved conditions. (Image credit: Smithsonian Institution Archives, NZP-0412) influence, designated and mapped as "anthromes" by Ellis et al. (2010).
The sample used by Eller et al. (2021)

| Skin color and dietary influences on MBD development
Diet and environment play an important role in mediating nutritional status. Recent evidence suggests that there are differences in initial cutaneous vitamin D metabolites between Papio species based on exposed facial skin pigmentation and sunlight conditions (Ziegler et al., 2018). A major limitation of the Ziegler et al. (2018) study was that UV exposure and diet were not controlled across the different baboon groups. Thus, our study examined whether differences in disease presentation between the six species were correlated with physiological differences associated with skin pigmentation. We found that this was not the case. While baboons display interspecies variability in skin pigmentation (Hill, 1967;Kamilar, 2006), this trait alone likely did not influence the presentation of MBD, which may instead reflect a combination of environmental factors.
Baboons may not be as reliant on sunlight exposure for vitamin D as other species, but rather are supplemented through dietary sources in their natural habitats. Most of their skin is covered in a thick pelage (regardless of color), which may hinder cutaneous vitamin D production. Further, they are known as hardy and diverse dietary generalists who can survive on a nonspecialized diet (Codron et al., 2006) and have been known to "raid" anthropogenic spaces for food items (Fehlmann et al., 2017). While some raids occur in more urbanized settings, such as Cape Town, South Africa (van Doorn & O'Riain, 2020), most are crop raids targeting small farming settlements across Africa (Hill, 2000;Kifle & Bekele, 2020;Maples et al., 1976;Warren, 2009 (Jasinghe & Perera, 2005), and P. cynocephalus at Mukumi National Park (Tanzania), for example, are known to utilize these food sources often (Norton et al., 1987). However, most mammals are unable to process vitamin D when it is in the form of vitamin D 2 and so mushrooms may not contribute much to the vitamin D requirements for baboons (Horst et al., 1990). Meat, particularly fish, fatty livers, and egg yolks, is the best source of dietary vitamin D aside from natural production via UV radiation (Schmid & Walther, 2013).

CONFLICT OF INTEREST
The authors declare no conflict of interest in the publication of this study.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available in the supporting information of this article and at OSF (https://doi.org/10.